Lysis of oncornaviruses by human serum. Isolation of the viral complement (C1) receptor and identification as p15E.

Moloney leukemia virus activated both the classical and alternative pathways of human complement. About 500,000 virions were required to detect activation of the classical pathway whereas 5,000 times as many virions were necessary to initiate the alternative pathway, indicating that in this system only the former is of biological significance. Disruption of the virus with Triton X-100 destroyed its ability to initiate the alternative pathway without affecting its ability to activate the classical pathway. After ultracentrifugation of disrupted virus the active component could be recovered in the supernate and was isolated by isoelectric focusing in granulated gels. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic and analysis and cyanogen bromide digestion studies revealed that the activity resided in a methionine-containing protein having a pI of 7.5 and a molecular weight of approximately equal to 15,000 daltons. The purified protein interacts strongly with Clq and efficiently activates Cl. RNase and lipolytic enzymes had no effect on the isolated protein but incubation with trypsin resulted in loss of activity. Enzymatic digestion studies of surface-labeled virus indicate that the active protein is a viral membrane protein. On the basis of these results it is concluded that the complement receptor of Moloney leukemia virus is the surface protein p15E.

Membrane attack complex of complement: a structural analysis of its assembly.

This study was conducted to gain insight into the process of assembly of the membrane attack complex (MAC) of complement through structural analysis. Four intermediate complexes and the MAC were examined by electron microscopy and by sucrose density-gradient ultracentrifugation. The C5b-6 complex has a sedimentation rate of 11S, an elongated, slightly curved shape and dimensions of 160 x 60 x 60 A. At protein concentrattions greater than 1 mg/ml, and physiologic ionic strength and pH, the complex forms paracrystals that have the appearance of parallel strands. Equimolar quantities of C5b-6 and C7 mixed in the absence of lipids or detergents give rise to C5b-7 protein micelles which are soluble in aqueous media and have a sedimentation rate of 36S, suggesting a tetrameric composition. Ultrastructurally, C5b-7 protein micelles consist of four half-rings, each measuring 200 x 50 A, which are connected to one another by short stalks extending from the convex side of the half-rings. C5b-7 bound to dioleoyl lecithin (DOL) vesicles has a similar ultrastructural appearance. After extraction with deoxycholate (DOC), C5b-7 has a sedimentation velocity of 36S which further suggests the occurrence of C5b-7 in the form of tetrameric protein micelles. Attachment of C8 to vesicle-bound C5b-7 results in dissociation of the protein micelles. An individual C5b-8 complex appears as a half-ring attached to the DOL-vesicle via a 100-A-long and 30-A-wide stalk. After extraction from the DOL-vesicles with DOC, C5b-8 has a sedimentation velocity of approximately 18S. Binding of C9 to DOL-vesicle bound C5b-8 induces the formation of the typical ultrastructural complement lesions. C5b-9 extracted from the vesicles with DOC has a sedimentation rate of 33S, which is characteristic of the C5b-9 dimer. It is concluded that dimerization is a function of C9. C5b-9 monomers are visualized when a single C5b-9 complex or an odd number of complexes were bound per DOL-vesicle. The C5b-9 monomer has an ultrastructural appearance that is theoretically expected of a half-dimer: a 200- x 50-A half-ring which is attached to the DOL-vesicle by a 100- x 80-A appendage. Extracted with DOC, the C5b-9 monomer has a sedimentation rate of 23S. At a higher multiplicity of MAC per DOL-vesicle, large structural defects in the lipid bilayer are seen which are attributed to direct physical destruction of membranes by the known lipid-binding capacity of the MAC. It is proposed that protein micelle formation at the C5b-7 stage of MAC assembly and dissociation of these micelles upon binding of C8 are events that facilitate dimerization of C5b-9 and thus MAC formation.

Occurrence of an incomplete C8 molecule in homozygous C8 deficiency in man.

Sera from unrelated individuals with recurrent Neisserial infections lacked C8 hemolytic activity, but contained a protein that is antigenically related to C8. Immunochemical analysis revealed complete identity of the C8-related protein of all three sera and a marked antigenic deficiency compared with normal C8. The C8-related protein was isolated from serum by adsorption to immobilized anti-C8 IgG, elution with 3 M guanidine, and subsequent gel filtration. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the abnormal protein resembled the alpha-gamma subunit of normal C8 with respect to mobility and its ability to be cleaved upon reduction into the alpha and gamma chains. The beta chain present in normal C8 was absent. Sedimentation equilibrium analysis indicated a molecular weight of 86,000 for the abnormal C8 protein, which is identical to that of the alpha-gamma subunit of normal C8. Amino acid analysis revealed no significant difference between the abnormal C8 and normal alpha-gamma. Unlike normal C8, the abnormal protein did not bind to EAC1-7 or to SC5b-7; however, upon addition to the deficient serum of beta chain isolated from normal C8, hemolytic activity was restored and formation of SC5b-9 occurred. We concluded that the dysfunctional C8 protein in the three individuals' serum is identical to the alpha-gamma subunit of normal C8 and that this form of C8 deficiency is distinct from the C8 deficiencies previously reported in which the entire three-chain protein is lacking.

Activation of complement by serum-resistant Neisseria gonorrhoeae. Assembly of the membrane attack complex without subsequent cell death.

Interaction of the human complement system in normal human serum (NHS) with serum-resistant and -sensitive Neisseria gonorrhoeae was evaluated to better understand the mechanism of serum-resistance. Complement activity (CH50) was depleted from NHS in a dose-dependent fashion by both serum-resistant and -sensitive N. gonorrhoeae. No detectable CH50 remained in NHS incubated with 10(9) colony-forming units (CFU)/ml serum of either resistant or sensitive strains. When smaller numbers of bacteria were incubated with NHS, lesser, yet comparable, amounts of CH50 were depleted by both resistant and sensitive strains. Hemolytic C2 activity was diminished by 33% in the case of resistant N. gonorrhoeae (10(8) CFU/ml serum) and by 48% in the case of a sensitive strain. No detectable decreases in hemolytic C4 or C7 activities were found with either sensitive or resistant strains at this concentration. Both resistant and sensitive strains activated C1s in NHS. Resistant strains specifically activated 19-21% of radiolabeled C1s in NHS, whereas sensitive strains activated 18-32%. Both resistant and sensitive strains also activated C5 in NHS. In binding assays using radiolabeled C5 and C9 in NHS, resistant and sensitive strains bound comparable amounts of C5 and C9. The number of bound C5 and C9 molecules varied according to the number of bacteria or amount of serum used in the assay. The ratio of C9/C5 bound to a sensitive strain was 6.8, and to a resistant strain was 8.2, suggesting that C5 and C9 were incorporated into membrane attack complexes (MAC). Electron microscopic examination of resistant and sensitive strains incubated with NHS revealed that MAC is bound to the surfaces of the resistant strain as well as the sensitive strain.

Horse complement protein C9: primary structure and cytotoxic activity.

Lack of hemolytic activity of horse serum is an inherent property of horse C9. To understand the molecular reasons for this deficiency we have cloned C9 cDNA from a horse liver cDNA library and have sequenced the cDNA yielding the complete coding sequence for horse C9. Purification of C9 from horse plasma and microsequencing established the N-terminus of the mature protein and verified that the correct horse C9 cDNA clone had been isolated. The deduced amino acid sequence corresponds to a mature protein of 526 amino acids that is 77% identical to human C9. It has the same domain structure as human C9 and contains 22 cysteines and four invariant tryptophans. The few differences include the N-terminus, which is an unblocked glycine in horse C9 but pyroglutamine in human C9, and three potential N-glycosylation sites compared to two in human C9. The N-terminal difference is unimportant since microsequencing of bovine C9, which is strongly hemolytic, established that it also has an unblocked glycine identical to horse C9. There are no obvious structural differences apparent that could resolve the differences in hemolytic potency between the two molecules. Aside from a few conservative replacements, both C9 sequences are identical between positions 250 and 360. This region includes the membrane interaction domain in C9 and the postulated transmembrane segment that is thought to constitute the wall of a putative transmembrane pore and, therefore, should be required for cytotoxicity. In agreement with this prediction we have observed that, in contrast to the marked decrease in hemolytic activity, horse C9 is very efficient in killing a variety of Gram-negative bacteria. These results demonstrate that horse C9 is a structurally competent molecule with efficient cytotoxic activity. Its inability to lyse erythrocytes may be related to the action of control proteins on target cell membranes.

Quantitation of the membrane attack complex of complement in an air-driven ultracentrifuge.

A sensitive assay of complement (C) activation via either the classical or alternative pathway was developed by evaluating assembly of the terminal complexes (C5b-9)2 or SC5b-9. Activation of serum containing [125I]C7 resulted in the formation of a stable, radiolabeled complex which was separable from its precursors by sedimentation in an air-driven ultracentrifuge. The radioactivity in the sediment was directly proportional to the amount of complex formed and assembly of the complex could be detected after C activation by aggregated IgG in concentrations as low as 10 micrograms/ml. Mild detergents such as Triton X-100 could be included in the reaction mixture, because they affected neither the assembly nor the integrity of the complexes. The assay, which detects both assembly of the membrane attack complex (MAC or (C5b-9)2) on target membranes and formation of SC5b-9 in fluid phase, measures the potential of certain substances to trigger the cytolytic phase of C regardless of whether the classical or alternative pathway was activated. However, by using serum depleted of either factor B or C1q, activation of either pathway can be assessed individually.

Domain structure, functional activity, and polymerization of trout complement protein C9.

The 3' region of trout C9 has been resequenced and found to differ from the previously published sequence (Stanley and Herz, EMBO J. 6:1951; 1987). In contrast to other sequenced C9 molecules, but in common with the other terminal complement components, trout C9 was found to contain an additional carboxy terminal thrombospondin domain. This domain does not restrict polymerization, as has been previously suggested (Stanley and Luzio, Nature 334:475; 1988), since alternative pathway activation of trout complement by rabbit erythrocytes lead to the formation of circular membrane attack complement lesions on the erythrocyte membrane. Although the trout C9 molecule is larger than human C9, the diameters of circular trout membrane attack complexes were approximately 30% smaller than their human counterparts. No lysis of erythrocytes bearing human C5b-7 or C5b-8 complexes was detected following incubation with trout serum containing EDTA, which suggests that trout C8 and C9 are unable to bind to human C7 and C8, respectively. Finally, trout and human serum were equally effective at killing the human serum-sensitive strain Salmonella minnesota Re595.

The membrane attack complex of complement. Assembly, structure and cytotoxic activity.

The membrane attack complex of complement is formed by the molecular fusion of the five terminal complement proteins, C5, C6, C7, C8, and C9. While the assembly process on a target membrane and its modulation by restriction factors present on host cells is now quite well understood the molecular details of the architecture of the complex still need much further clarification. This is especially true for the interaction of the last acting protein C9, which provides the cytotoxic action of the complex, with the precursor C5b-8 complex. Because of this lack of structural details the molecular mechanisms that lead to complement-mediated cell death remain cryptic, however, it is hoped that recent advances in controlling the assembly process and in site-specific modification of the terminal complement proteins by recombinant DNA techniques should change this predicament quickly.

Effects of drug-binding on the thermal denaturation of human serum albumin.

Asymmetric thermograms of defatted albumin, alone and in the presence of two model drugs, have been obtained in phosphate buffers at three pH values. The albumin is less thermally stable in the N form, but is protected by both drugs. The nonsteroidal antiinflammatory benoxaprofen offers more protection than warfarin against thermal denaturation.

Thermal unfolding and aggregation of human complement protein C9: a differential scanning calorimetry study.

The thermotropic behavior of purified human complement protein C9 was investigated by high-sensitivity differential scanning calorimetry. When dissolved in physiological buffers (pH 7.2, 150 mM NaCl), C9 underwent three endothermic transitions with transition temperatures (Tm) centered at about 32, 48, and 53 degrees C, respectively, and one exothermic transition above 64 degrees C that correlated with protein aggregation. The associated calorimetric enthalpies of the three endothermic transitions were about 45, 60, and 161 kcal/mol with cooperative ratios (delta Hcal/delta HvH) close to unity. The total calorimetric enthalphy for the unfolding process was in the range of 260-280 kcal/mol under all conditions. The exothermic aggregation temperature was strongly pH dependent, changing from 60 degrees C at pH 6.6 to 81.4 degrees C at pH 8.0, whereas none of the three endothermic transitions was significantly affected by pH changes. They were, however, sensitive to addition of calcium ions; most affected was Tm1 which shifted from 32 to 35.8 degrees C in the presence of 3 mM calcium, i.e., the normal blood concentration. Kosmotropic ions stabilized the protein by shifting the endothermic transitions to slightly higher temperatures whereas inclusion of chaotropic ions (such as choline), removal of bound calcium by addition of EDTA, or proteolysis with thrombin lowered the transition temperatures. Previous studies had indicated the formation of at least three different forms of C9 during membrane insertion or during heat polymerization, and it is suggested that the three endothermic transitions reflect the formation of such C9 conformers. Choline, which is present at high concentrations on the surface of biological membranes, and calcium ions have the ability to shift the transition temperatures of the first two transitions to be either close to or below body temperature. Thus, it is very likely that C9 is present in vivo in a partially unfolded state when bound to a membrane surface, and we propose that this facilitates membrane insertion and refolding of the protein into an amphiphilic conformation.

Complement-mediated killing of Escherichia coli: dissipation of membrane potential by a C9-derived peptide.

The molecular mechanism of complement-mediated killing of Gram-negative bacteria has yet to be resolved, but it is generally accepted that assembly of the membrane attack complex (MAC) of complement on the outer bacterial membrane is a required step. We have now investigated the effect of the MAC and its precursor complex, C5b-8, on the membrane potential (delta Em) across the inner bacterial membrane. Delta Em of whole cells was measured directly by using a lipophilic cation (tetraphenylphosphonium) that equilibrates with the potential or indirectly by measuring transport of solutes (proline and galactoside), which is dependent on delta Em. Our results indicate that the C5b-8 complex caused a transient collapse of delta Em in the absence of cell killing. Addition of C9 to allow formation of the MAC dissipated delta Em irreversibly, and the cells were killed. Since delta Em is generated across the inner membrane in Gram-negative bacteria, inner membrane vesicles were prepared and membrane potentials were generated either by adding D-lactate to energize the electron-transport chain or by creating a K+ diffusion potential with valinomycin. C9 added in the absence of earlier acting complement proteins had no effect on delta Em of isolated, actively respiring vesicles or on K+ diffusion potentials. In contrast, its C-terminal thrombin fragment (C9b), which has been shown earlier to contain the membrane-active domain of C9, efficiently collapsed delta Em in such vesicles. C9b did not require a specific receptor since it was effective on "right-side-out" and "inside-out" vesicles. These results are interpreted to indicate that a C9-derived fragment deenergizes cells and may be the causative agent for cell death.

Identification of the discontinuous epitope in human complement protein C9 recognized by anti-melittin antibodies.

Polyclonal rabbit antibodies against melittin recognize human C protein C9 and retard C9-mediated hemolysis. Human C9 contains a tetrameric and a pentameric sequence (amino acids 293-296 and 528-532, respectively) that together match a continuous segment in the melittin sequence, i.e., residues 8-16. It has been suggested that the tetrameric and the pentameric regions on C9 form a discontinuous epitope on folded C9 that mimics the structure of melittin. To further test this hypothesis, antibodies to C9-sequence-specific peptides were prepared. Peptides containing either the homologous tetrameric or the homologous pentameric sequence together with short stretches of the respective amino- and carboxyl-terminal flanking regions were synthesized, as well as a composite peptide predicted to resemble the discontinuous epitope as a linear, nine-amino acid sequence. Direct and competitive binding assays demonstrated that the tetrameric and the pentameric sequences are part of the epitope on human C9 that is recognized by anti-melittin IgG. However, only antibodies directed against the complete epitope are capable of inhibiting hemolysis. Because neither anti-tetramer nor anti-pentamer antibodies affect hemolysis whereas anti-melittin and anti-composite antibodies do, we propose that human C9 changes conformation around a hinge located between residues 296 and 528 and that the latter two antibodies inhibit unfolding required for membrane insertion and subsequent hemolysis.

Detection of refolding conformers of complement protein C9 during insertion into membranes.

Human complement protein C9 is a hydrophilic serum glycoprotein responsible for efficient expression of the cytotoxic and cytolytic functions of complement. It assembles on the surface of a target cell together with C5, C6, C7 and C8 to form the membrane attack complex (MAC) and therefore has to change structure to become an integral membrane protein. As the protein assumes a stable structure in an aqueous environment, the question arises as to how it can enter the hydrophobic interior of a membrane. During MAC assembly C9 polymerizes into a circular structure, termed poly(C9) (ref. 8), which is responsible for the cylindrical electron microscopic appearance of the MAC. The suggestion has been made that C9 must at least partly unfold in order to enter a membrane and also that polymerization of the molecule is intimately linked to insertion and cytotoxicity. The extent of unfolding and the mechanism of polymerization are not understood, nor is it known precisely which parts of the molecule participate in the proposed structural changes. We have been able to capture refolding C9 conformers during membrane insertion with the help of sequence-specific anti-peptide antibodies. Some of these antibodies inhibit C9-mediated haemolysis but not C9 polymerization, while others have the opposite effect. This suggests that the two processes are independent.

Bacterial killing by complement. C9-mediated killing in the absence of C5b-8.

The ability of serum complement to kill Gram-negative bacteria requires assembly of the membrane attack complex (MAC) on the cell surface. The molecular events that lead to cell killing after MAC assembly are unknown. We have investigated the effect of C9 on bacterial survival in the presence and absence of its receptor, the C5b-8 complex, on the outer membrane. A fluorescence assay of the membrane potential across the inner bacterial membrane revealed that addition of C9 to cells bearing the performed C5b-8 complex caused a rapid and complete dissipation of the membrane potential. No fluorescence change was observed in serum-resistant strains of Escherichia coli. Addition of trypsin, after C9 was bound to C5b-8, did not rescue the cells from the lethal effects of C9. Furthermore, assays of cell killing kinetics and C9 binding indicate that formation of tubular poly(C9) is not required for killing. When C9 was introduced into the periplasmic space in the absence of its receptor by means of an osmotic shock procedure, cell killing occurred. Other proteins, such as C8 or serum albumin, were not toxic, and C9 was ineffective against two resistant strains. The results presented here and previously [Dankert & Esser (1986) Biochemistry 25, 1094-1100], when considered together, indicate that the 'lethal unit' in complement killing of some Gram-negative bacteria is a C9-derived product that acts by dissipation of cellular energy.

Mechanism of antibody-independent activation of the first component of complement (Cl) on retrovirus membranes.

Murine leukemia viruses activate human C1 in the absence of specific antibody. Such activation requires the binding of C1 to the viral surface through two subcomponents, C1q and C1s. This conclusion is based on the following results. (1) Isolated human C1q and C1s bind the same membrane protein on virions. (2) Binding one subcomponent is independent of the other. (3) Only dimeric C1s binds, whereas monomeric C1s, prepared by dissociation with ethylenediaminetetraacetate (EDTA), has no affinity for the virus. (4) The activated C1s dimer, C1s, does not attach to the virus. (5) Saturation of C1s binding sites on the viral surface does not prevent binding of macromolecular C1, but such bound C1 is not activated. (6) No exchange occurs between C1s bound to the viral membrane and C1s contained in C1, which in turn is attached via C1q to the same virus. Therefore activation occurs only when both C1q and C1s in the same C1 complex in contact with the viral activator. Human C1r has no affinity for the virus nor does guinea pig C1s. The latter result explains why guinea pig serum does not function in antibody-independent virolysis.

The first complement component: evidence for an equilibrium between C1s free in serum and C1s bound in the C1 complex.

An equilibrium between free C1s and C1s bound in macromolecular C1 exists in human serum. This equilibrium can be utilized to incorporate radioiodinated C1s into serum C1. Human sera were incubated for 40 hr at 4 degrees C with 125I-C1s to allow the exchange between free and bound C1s to reach equilibrium. The C1 complex labeled in this manner was separated from the majority of serum proteins by centrifugation in linear 10 to 30% sucrose density gradients. The resulting fractions containing 125I-C1 can be used directly and conveniently in C1 activation assays that detect the cleavage of proenzyme C1s. Electrophoretic analysis on polyacrylamide gels showed the presence of only proenzyme 125I-C1s in serum C1, whether or not the applied labeled material contained 125I-C1s or other labeled proteins. The inability of C1 to incorporate C1s was shown to be the result of decreased stability of C1 upon activation.